Quartz Crystal Microbalance Application and In Silico Studies to Characterize the Interaction of Bovine Serum Albumin with Plasma Polymerized Pyrrole Surfaces: Implications for the Development of Biomaterials

Plasma polymerized pyrrole/iodine (PPPy/I) microparticles and bovine serum albumin (BSA) protein have shown interesting results in experimental models for the treatment of traumatic spinal cord injury. By studying the interaction between BSA and PPPy/I by a quartz crystal microbalance (QCM) and docking, we obtained important results to elucidate possible cellular interactions and promote the use of these polymers as biomaterials. These measurements were also used to characterize the adsorption process using an equilibrium constant. In addition, atomic force microscopy (AFM) was used to obtain images of the QCM surface sensors before and after BSA adsorption. Furthermore, we carried out molecular dynamics simulations and molecular docking to characterize the molecular recognition between BSA and the previously reported PPPy/I structure. For this study, we used two combinatorial models that have not been tested. Thus, we could determine the electrostatic (ΔGele) and nonelectrostatic (ΔGnonelec) components of the free binding energy (ΔGb). We demonstrated that BSA is adsorbed on PPPy/I with an adsorption constant of K = 24.35 μ–1 indicating high affinity. This observation combined with molecular docking and binding free energy calculations showed that the interaction between BSA and both combinatorial models of the PPPy structure is spontaneous.


Dynamic light scattering of BSA protein
Figure S1.DLS from the BSA protein.

Physicochemical characterization of PPPy/I films
Plasma polymerized polypyrrole (PPPy) is a complex polymer with average characteristics that are reproducible, although its composition might change depending on the synthesis variables (power, pressure, reaction time).The synthesis conditions used in this work have already been reported. 1for convenience we add this additional summary with the basic molecular and surface characterization: Fourier Transform Infrared (FTIR), X-ray Photonic Spectroscopy (XPS) and Contact Angle (CA).

FTIR spectrum
Figure S2 presents the FTIR spectrum of the PPPy/I, the region between 3500 and 3300 cm - 1 shows a broad band with the NH and OH groups.In the case of nitrogen band, the group can belong to primary or secondary amines.Between 2960 and 2850 cm -1 we have alymphatic CH.In the frequency interval 2150-2260 cm -1 there is a band that can be assigned to vibrations of C≡N and C≡C.These last two bands are not found in the chemically synthesized polypyrrole, which gives evidence of monomer cleavage during plasma polymerization.In 1593 cm -1 we have a band that can be associated with amine groups and C=C groups associated with the pyrrole ring, in 1495 we have the C-N groups, this resonance as well as that of 1250 cm -1 appear at these frequencies because they are associated with the pyrrole aromatic ring.

Computational studies of human serum albumin (HSA) and PPPy combinatorial models.
Figure S6.Exhaustive study of molecular dynamics simulations of HSA and its interaction with PPPy combinatorial models by molecular docking assays and binding energy calculations.
Human albumin (PDB ID 5YB1) 3 was used as initial structure for molecular dynamics simulations.5][6] All hydrogens were used and a 0.15 mM of KCl concentration was used.Periodic Boundary conditions were placed and PME for electrostatics were used as indicated on CHARMM-GUI server.GROMACS input was used.CHARMM 36m potential 7,8 was used on GROMACS 2019. 9-11A 2 fs integration time step was used for 100 ns.Three independent 100 ns molecular dynamics simulations were conducted.Root mean square deviation, distances and cluster analysis were conducted with the tools included on GROMACS.
Molecular docking was conducted using Autodock Vina. 12100 independent blind molecular docking experiments were conducted on each cluster structure (one for each system).All outputs were root mean square deviations (RMSD) clustered and the most populated one is herein presented.Images were produced using VMD 1.9.3 13 and Chimera UCSF. 14,15 conducted three independent 100 ns molecular dynamics simulations on human albumin.Figure S6.1A illustrates the temporal fluctuations observed in the α-carbon RMSD for the entire protein over the 100 ns simulated against the initial structure.As noted, all systems appeared to be fluctuating along the simulated time.To pinpoint the specific region responsible for these RMSD fluctuations, we focused our analysis on the final 40 ns of each simulation.Upon analyzing residues 3 to 300 (Figure S6.1B on blue) and 301 to 583 (Figure S6.1B on orange), we discovered that both sections exhibited remarkable stability throughout the analyzed time (Figure S6.1C).
Consequently, we concluded that the molecular dynamics simulations remained stable during the last 40 ns, and the observed RMSD fluctuations were attributed to movements in both sections of the protein.Based on the results obtained, we look for a representative structure of the last 40 ns of each simulated system.A representative structure (the most populated cluster contains at least 70% of the structures analyzed) of the last 40 ns were obtained for each system.Figure S6.3 shows the superposition between each cluster found against the initial structure.Each structure was used as receptor for a blind docking with modified PPPy ligand with two combinatorial compounds CC 1 (R1=OH, R2=CH3, R3=NH2); and CC 2 (R1=NH2, R2=CH3, R3=OH).Molecular affinity energies for the most populated cluster are shown in Table S1.S1, all affinities energies were similar.These results showed that CC 1 and CC 2 may bound albumin with similar affinities independent of the protein conformation.
To determine the position of both combinatorial compounds on the albumin surface we obtained the interaction maps between the ligands and the protein.Subsequently, we calculated the Gb for each combinatorial model of the modified PPPy structure on the representative structure of HSA of the most populated clusters of each molecular dynamics, we considered the electrostatic and non-electrostatic components as described by Baker et al.As observed, the HSA Gb values changed in all the molecular dynamics simulations.However, we showed that the trend in both HSA and BSA was preserved.As with BSA, the HSA interaction for CC 1 (R1=OH, R2=CH3, R3=NH2) of the modified PPPy structure, the Gb values were more favorable compared to CC 2 (R1=NH2, R2=CH3 and R3=OH).Furthermore, the binding is governed by the Coulombic component in both cases (HSA and BSA).Also, the binding free energy results are complementary with docking studies because CC 1 established more hydrogen bonds compared to CC 2 mainly when the functional group NH2 is in the longest chain of the modified PPPy structure compared to the group OH, and this aspect plays a very important role in the protein interaction, which in consequence was reflected in the Gb values.On the other hand, in the Cluster C both CC 1 and CC 2 established hydrophobic interactions, hence the electrostatic component was not favored and therefore the G>0 values.

Figure S6. 1 .
Figure S6.1.A) α-carbons RMSD over 100 ns of each system simulated.B) Albumin segments used to calculate the RMSD (blue, residue 3 to 300 and orange, residues 301-583).C) RMSD over the last 40 ns of each system showing the RMSD for all the system and by segment.

Figure S6. 2 .
Figure S6.2.Distance of the center of mass of the two domains herein used (3-300 and 301-583).Color code are the same as Figure S6.1A (System A on black, B on red and C on green).

Figure S6. 3 .
Figure S6.3.Cartoon representation of each cluster found for each system (System A on blue, B on red and C on green) against initial structure.
Interaction maps are found in Figure S6.4.

Figure S6. 4 .
Figure S6.4.Interaction maps between Combinatorial Compound 1 and 2 against each cluster found from molecular dynamics.Cluster A (left), B (center) and C (right) for A) Combinatorial Compound A and B) Combinatorial Compound B.

Table S1 .
Affinities Energies between each cluster found from albumin molecular dynamics and combinatorial compounds (CC). 16

Table S2 .
Gb values for the representative structure of HSA of the most populated clusters of each

Table S3 .
Gb values for the representative structure of HSA of the most populated clusters of each molecular dynamics with the CC 2 (R1=NH2, R2=CH3, R3=OH) of the modified PPPy structure determined at pH 7.2 by APBS and VMD1.9.1.